Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle that alternates between mammals and a tick vector. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual segmented genome that includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded genes are critical for successful adaptation by B. burgdorferi to the different environments that the spirochete encounters during its infectious cycle. A major focus of this project is to determine how and why the Lyme disease spirochete maintains such a unique genomic structure and the specific contributions of individual plasmids and genes at each stage of the infectious cycle. We have addressed this topic primarily through a molecular genetic approach. The genome of B. burgdorferi consists of a small linear chromosome and more than 20 linear and circular plasmids. Although some plasmids are required by B. burgdorferi for growth in its natural hosts, most plasmids are dispensable for growth in culture medium. However, one plasmid, cp26, is present in all natural B. burgdorferi isolates and has never been lost during propagation of the spirochete in the laboratory. We previously demonstrated that cp26 encodes essential functions required by B. burgdorferi for growth under all conditions and hence cannot be lost or actively displaced from viable organisms. In FY2009 we established that the guaAB genes on cp26, while not required for B. burgdorferi growth in culture medium, are critical for spirochete survival in an experimental model system that replicates the natural infectious cycle (Jewett et al. J. Bacteriol in press 2009). The guaA and guaB genes encode GMP synthase and IMP dehydrogenase (IMPDH) respectively, which are key enzymes in purine salvage and guanine nucleotide biosynthesis. IMPDH activity (encoded by guaB) is critical for the survival of most organisms. Using a molecular genetic approach, we demonstrated that the enzymatic activities encoded by the guaAB operon are required by B. burgdorferi to colonize the mammalian host and important for spirochete replication in the tick vector (Jewett et al. J. Bacteriol in press 2009). We determined that the guaAB operon is transcribed throughout infection, an expression pattern that contrasts sharply with that of the ospC gene, which is upstream and divergently transcribed from the guaAB locus. OspC is an outer surface lipoprotein that is synthesized during the initial stage of mammalian infection, but subsequently down regulated in order to evade the acquired immune response of the host. Thus, although guaAB and ospC share an upstream intergenic region that harbors regulatory sequences critical for ospC repression, the guaAB operon is not co-regulated by these DNA sequences. While cp26 carries genes like guaAB that provide critical metabolic functions, we have previously shown that OspC is a virulence factor that is absolutely and exclusively required by B. burgdorferi for the initial colonization of the mammalian host, following either tick bite or needle inoculation. In FY2009, to address the role of OspC in the early steps of B. burgdorferi infection of mammals, we used skin from persistently infected mice to infect nave mice (Tilly et al, Infection and Immunity, 77:2672-2682, 2009). Since OspC is not required by B. burgdorferi subsequent to the initial stage of infection, we have previously shown that it is possible to derive mice persistently infected with ospC mutant spirochetes that only transiently contained an unstable wild-type copy of the ospC gene. We transferred skin from mice carrying wild-type and ospC mutant spirochetes to nave mice and found that both were equally capable of establishing a disseminated infection by this route. We concluded that OspC, whose production is absolutely essential for infection by tick-bite and needle inoculation, is dispensable for infection by transfer of host-adapted spirochetes. We propose that the essential function typically fulfilled by OspC during the initial stage of infection is required throughout infection, but another protein assumes that function during persistence and is present on host-adapted spirochetes in the skin. The essential nature of OspC for colonization of mice by B. burgdorferi preceding the acquired immune response suggests a critical role for OspC in evasion of host innate immunity. Microorganisms induce a variety of responses from the skin of their host, including antimicrobial peptides like defensins and cathelicidins, which are integral components of the innate immune system. The importance of murine cathelicidin, known as mCRAMP, to innate host defense is well established. Unlike many bacterial pathogens, B. burgdorferi is highly resistant to cathelicidin, consistent with the spirochetes ability to persistently colonize the skin, where antimicrobial peptides are present. We hypothesized that as an abundant surface protein with limited membrane contact, OspC could initially shield the spirochete from lytic components of innate defense like cathelicidin. This potential role for OspC in resistance to antimicrobial peptides is consistent with our previous demonstration of the rapid clearance from the skin of OspC mutants. In FY2009, we analyzed the cathelicidin resistance of B. burgdorferi variants that differed in outer surface lipoprotein composition (Sarkar et al, Antimicrob. Agents Chemother., in press 2009). We compared the survival, following incubation with mCRAMP, of strains synthesizing or lacking OspC. All strains were highly resistant to killing at a wide range of antimicrobial peptide concentration, irrespective of their OspC phenotype. OspA is an abundant surface lipoprotein that is synthesized by spirochetes during in vitro propagation, but which is typically not made in the mammalian environment. To determine whether the presence of OspA on in vitro grown spirochetes contributed to the observed cathelicidin resistance and masked an otherwise critical role of OspC, we constructed and tested a double mutant that lacks OspA and OspC (Sarkar et al, Antimicrob. Agents Chemother. in press 2009). However, we found that this strain still maintained a high level of cathelicidin resistance. To determine whether sensitivity to cathelicidin reflects peptide binding to the outer surface of the spirochete, we assessed mCRAMP binding to B. burgdorferi. All strains, regardless of the presence of OspA, OspC or other lipoproteins, exhibited limited mCRAMP binding. We conclude that the critical early role of OspC during mammalian infection is unrelated to resistance to major antimicrobial peptides in the skin. Our results support a model by which the observed resistance of B. burgdorferi to cathelicidin correlates with a lack of antimicrobial peptide binding and we hypothesize that this characteristic is consistent with the unique biophysical properties of the Borrelia outer membrane, independent of lipoprotein content.